1. Field of the Invention
The present invention provides an avian recombinant herpesvirus comprising cDNA of VP2 (VP2 cDNA) of the IBDV Delaware Variant E strain, a member of IBDV variant strain subtype.
2. Related Art
Infectious Bursal Disease, often referred to as Gumboro disease, is caused by the highly transmissive Infectious Bursal Disease Virus (IBDV) and inflicts a great economic loss on the poultry industry. There are two serotypes of IBDV: serotype 1 and 2. Serotype 1 is pathogenic for chickens. Many IBDV strains are classified as serotype 1. Based on pathogenicity and antigenicity, serotype 1 strains are further divided into four subtypes: classical virulent, attenuated, variant, and very virulent strains (Y. C. Cao et al., 1998, Avian Diseases, 42: 340-351). For instance, STC, 52/70, 002-73, etc., are classified as classical virulent strains. Bursine, Bursa-Vac-3, Bursa-Vac-M, CU-1, PBG98, etc., are classified as attenuated strains. Delaware E, A, GLS, GZ902, Ark, AL-2, etc., are classified as variant strains, and OKYM, UK661, F9502, HK46, etc., are classified as very virulent strains.
The IBDV genome consists of two double-stranded RNA segments: Segment A and B. Segment A encodes a 115 kd precursor polyprotein, which is processed auto-catalytically by VP4 to give VP2, VP3 and VP4. Segment A also encodes VP5 that is translated in a different reading-frame from VP2 to VP4. Reportedly, VP5 is related to the virulence of the virus but its detailed function remains to be elucidated. Earlier studies using anti-IBDV monoclonal antibodies indicate that VP3 has one serotype-specific epitope and another non-overlapping epitope, but VP3 does not contain a major virus-neutralizing epitope. On the contrary, VP2 has independent epitopes that elicit IBDV neutralizing antibodies (U.S. Pat. Nos. 5,350,575, 5,849,575). The amino acid sequence of VP2 differs from strain to strain (H. G. Heine et al., 1991, J. Gen. Virol. 72: 1835-1843, T. V. Dormitorio et al., 1997, Avian Diseases 41:36-44, and Y. C. Cao, et al., 1998, Avian Diseases 42: 340-351), and antigenic differences among IBDV subtypes are likely due to these sequence variances.
In the United States, Infectious Bursal Disease has been controlled by passive immunity passed from the hen to the chick. In short, high antibody levels are elicited using killed IBDV vaccines so that chicks acquire high maternal antibody levels. These high maternal antibody levels protect the chick through the first few weeks of life. Problems associated with this strategy are that all chicks do not acquire the same level of maternal antibody. On a flock basis it is hard to predict when maternal antibodies wane and as a result some chicks are unprotected. In the United States in the 1980s, variant IBD viruses breaking through passive immunity elicited with classic vaccine strains caused disease in the United States (H. G. Heine et al., 1991, J. Gen. Virol. 72:1835-1843). The most prominent variant virus at this time was Delaware Variant E. Variant IBDV viruses were and are still being added to killed vaccines for protection against variant strains. In Europe, many cases of Infectious Bursal Disease were reported among chickens that had high titers of IBDV maternal antibody. These chickens were killed by the natural infection of a very virulent strain even though hens were administered IBD vaccines (M. D. Brown et al., 1994, J. Gen. Virol. 75:675-680). These incidents indicate that antigenic differences between vaccine and prevalent disease-causing strains should be seriously considered. Development of a vaccine that protects chickens from a variety of different subtypes of IBDV is desirable for the poultry industry.
Construction of a recombinant avian herpesvirus harboring a protective antigen gene from other avian pathogens as well as its use as a poultry vaccine is suggested in U.S. Pat. Nos. 5,834,305, 5,853,733, 5,928,648, 5,961,982, WO 87/04463 and WO 99/18215 etc. VP2 is a protective antigen of IBDV and Segment A of IBDV includes VP2 gene. The recombinant avian herpesvirus comprised of the VP2 gene or Segment A and its use as an IBD vaccine is reported in U.S. Pat. No. 5,733,554, WO 89/01040, WO 93/25665, WO 96/05291 or WO99/18215. R. Darteil et al., and K. Tsukamoto et al. also reported similar recombinant avian herpesvirus-vectored IBD vaccines (R. Darteil et al., 1995, Virology, 211:481-490, K. Tsukamoto et al., 1999, Virology, 257:352-362). In WO 89/01040, under the control of the pseudorabies virus gpX promoter, cDNA of Segment A including VP2, VP3 and VP4 genes was inserted into the BamHI #16 fragment in the UL43 gene of herpesvirus of turkeys (HVT) to generate a recombinant herpesvirus, S-HVT-003. Segment A was derived from the IBDV S4047 strain but its subtype is not divulged in the specification. In addition, R. Darteil et al. (R. Darteil et al., 1995, Virology, 211:481-490, U.S. Pat. No. 5,733,554) reported a few recombinant HVTs harboring VP2 gene from the IBDV 52/70 strain, a member of the classical virulent strain subtype. For instance, vHVT1 comprises the said VP2 gene in the RR2 (UL40) region, which is driven by the RR2 intrinsic promoter. vHVT2 comprises the VP2 gene under the control of the exogenous CMV-IE promoter in the gI (US7) region. vHVT4 comprises the VP2 gene driven by the same promoter in the UL43 region. Although the UL40 and US7 regions seemed not to be essential for in vitro virus growth, vHVT1 and vHVT2 did not grow well in vivo. On the contrary, vHVT4 conferred good protection in SPF chickens against challenge with the IBDV 52/70 strain. However, in this experiment, the challenge conditions seem to have been mild since the group of positive control chickens, vaccinated with an inactivated IBDV vaccine, was also completely protected. Inactivated vaccines do not induce protective immunity against very virulent strains or European types of virulent strains.
In addition, WO 99/18215 describes a recombinant HVT, HF003, which has the VP2 gene inserted into the inter-ORF region between UL45 and UL46. The said VP2 gene was from IBDV OKYM, a member of the very virulent strain subtype, which was isolated in Japan. However, HF003 was proven to confer protection only against the IBDV OKYM strain.
In consequence, several avian recombinant herpesviruses comprising IBDV genes have been reported so far, but none of these induced in chickens protective immunity against a variety of different subtypes of IBDV. In other words, no knowledge is available as to which VP2 gene is suitable for the construction of the recombinant avian herpesvirus that will give protection against the broad range of IBDV subtypes.
The present invention provides an avian recombinant herpes virus modified by the presence of the cDNA encoding the VP2 gene of the Delaware Variant E strain of IBDV. In chickens, the recombinant virus elicited excellent protective immunity against a variety of different IBDV strains belonging to two subtypes of serotype 1.
More specifically, the present invention provides an avian recombinant herpesvirus modified by the insertion of cDNA of the VP2 gene that is derived from Delaware Variant E, a member of the IBDV variant strain subtype. The insertion site of the VP2 cDNA is in a region non-essential for the avian herpesvirus growth (the non-essential region). The present invention further provides an Infectious Bursal Disease vaccine including the said avian recombinant herpesvirus as an active ingredient.
The present invention is described below in more details.
VP2 cDNA
As well as being derived from the Delaware Variant E strain, any VP2 cDNA can be used for the purpose of the present invention.
The amino acid sequence of VP2 differs from strain to strain (T. V. Dormitorio et al., 1997, Avian Diseases 41:36-44, Y. C. Cao, et al., 1998, Avian Diseases 42: 340-351.), suggesting that even among variant strains, the nucleotide sequence of VP2 cDNA may differ.
The nucleotide sequence of the VP2 cDNA of the Delaware Variant E strain is reported in the literature (H. G. Heine et al., 1991, J. Gen. Virol. 72: 1835-1843, European Molecular Biology Laboratory (EMBL) database (Accession# AF133904)). In addition, the sequence shown in SEQ ID No.1 is also representing VP2 cDNA because the VP2 gene sequence slightly differs from clone to clone. VP2 cDNA shown in SEQ ID NO.1 is, therefore, only an example that is suitable for the purpose of the present invention.
VP2 cDNA derived from the virus genome can be used with or without modifications to generate the avian recombinant herpesvirus. For instance as shown in SEQ ID NO.1, the VP2 cDNA can be modified to have a stop signal following the 453rd amino acid codon and restriction sites at both N- and C-terminals to facilitate its subcloning into the plasmid vector.
Promoter
As long as being functional in the avian herpesvirus-infected cells, any promoter can be used to express the inserted VP2 gene in the present invention. For instances, the exogenous promoter such as Cytomegalovirus (CMV) promoter, Rouse Sarcoma virus (RSV) promoter, SV40 early promoter, endogenous promoter such as MDV-1 gB promoter (U.S. Pat. No. 6,013,261) and the chicken beta-actin promoter are suitable. Among these, the chicken beta-action promoter is most favorable because it facilitates the high expression of the VP2 gene.
The nucleotide sequence of the chicken beta-action promoter is reported in the literature (T. A. Kost et al., 1983, Nucleic Acids Res. 11:8287-8301). However, the promoter for the present invention need not necessarily be identical to that reported. In fact, as shown in SEQ ID NO. 3, the beta-actin promoter cloned from chicken cells by the inventors had a slightly different sequence from that reported.
Addition of Other Nucleotides to VP2 cDNA
To stabilize the transcribed mRNA, the polyA signal can be added at 3xe2x80x2 terminus of the VP2 gene. Any polyA signal that enhances Eukaryotic gene expression is appropriate. An example is the SV40 polyA signal, which is included in pBK-RSV (STRATAGENE, Cat #212210).
Avian Herpesvirus
Any of serotype 1, serotype 2 or serotype 3 Marek""s disease virus can be used as a backbone avian herpesvirus. However, taking their use as the poultry vaccine into consideration, Marek""s disease vaccine strains such as HVT FC126 (serotype 3), SB1 (serotype 2) or Rispens (serotype 1) are suitable for the purpose of the present invention.
Non-essential Regions for the Gene Insertion
There are many reports of the non-essential regions of the avian herpesvirus, a non-essential region being dispensable for the virus growth and suitable for foreign gene insertion. VP2 cDNA can be inserted into any of these regions. For instance, the UL43 gene described in WO 89/01040, the US2 in WO 93/25665 and the inter-ORF region between UL44 and UL46 in WO 99/18215 can be used for the VP2 cDNA insertion. Among these, the inter-ORF region between UL44 and UL46 is most suitable in regard of the virus stability.
For the present invention, the non-essential region can newly be identified by the following general procedures. First, the avian herpesvirus DNA fragments of the appropriate length are cloned into an E. coli plasmid and physically mapped by restriction enzyme analysis. Then, a gene cassette consisting of a promoter and a marker gene is inserted into an appropriate restriction site of the cloned DNA fragment. As described later, if the homologous recombination with the resultant homology vector resulted in a recombinant virus expressing the inserted marker gene and if it is stable in vitro and in vivo, the originally selected DNA fragment should be a non-essential region suitable for VP2 cDNA insertion. To check the stability, the genome DNA of recombinant viruses was propagated on the marrow of purification, and after five more passages in vitro and in vivo was prepared and subjected to Southern hydridization analysis. If the result of the analysis indicates that the genome structures before and after passaging are identical, the recombinant virus is stable.
Construction of the Avian Recombinant Herpesvirus
For the present invention, any known method of generating the recombinant avian herpesvirus is applicable. A typical example is as follows. (1) First, as described above, a recombinant plasmid is constructed, which includes a non-essential region of the avian herpesvirus. Then, preferably with a promoter at the 5xe2x80x2 terminus and a polyA signal at the 3xe2x80x2 terminus, VP2 cDNA is inserted into the said non-essential region to generate a homology vector. (2) The resultant vector is transfected into chicken embryo fibroblast (CEF) cells infected with parent avian herpesvirus or co-transfected into CEF cells with infectious avian herpesvirus genomic DNA. Transfection is performed by any known method. (3) The transfected CEF cells are inoculated into culture plates and incubated till the virus plaques become visible. (4) The identifiable plaques include recombinant viruses as well as parent wild-type viruses. The recombinant virus is purified from these plaques by any known method. For instance, CEF cells having plaques are diluted to an appropriate concentration, transferred to the 96-well plates and recombinant plaques are selected by antigen-antibody reaction using the monoclonal antibody against the IBDV VP2 as the primary antibody.
Infectious Bursal Disease Vaccine
The recombinant avian herpesvirus in the present invention is used as a chicken Infectious Bursal Disease vaccine since it includes the VP2 gene. VP2 is a protective antigen of IBDV. a causative virus of Infectious Bursal Disease. In addition, when Marek""s disease virus (serotype 1, serotype 2 or serotype 3 MDV) is used as a backbone virus for VP2 gene insertion, it can be a polyvalent vaccine against both Infectious Bursal and Marek""s diseases.
For the purpose of the present invention, the poultry vaccine consisting mainly of the recombinant avian herpesvirus may include chicken cells and/or ingredients of culture media in addition to the serotype 1, serotype 2 or serotype 3 recombinant MDV. The vaccine may contain other ingredients such as preservatives, so long as these are not pharmacologically detrimental.
The poultry vaccine of the present invention may be combined with any recombinant or non-recombinant viruses. For example, serotype 1 or serotype 2 MDV vaccine strain can be mixed with the vaccine consisting mainly of the serotype 3 recombinant MDV.
Any known method is applicable to the preparation of the poultry recombinant polyvalent vaccine of the present invention. For instance, the recombinant MDV of the present invention is inoculated into permissive culture cells such as CEF cells and grown to an appropriate titer. Then, the cells are scraped off from culture plates or bottles by scraper or by trypsin treatment and subjected to centrifugation. Cells separated from the supernatant are then suspended in culture medium containing 10% dimethyl sulfoxide and stored in liquid nitrogen.
The avian polyvalent vaccine is administered to chickens by any known method of inoculating the Marek""s disease vaccine. For instance, the vaccine of the present invention is suspended in the phosphate buffer saline to give 10-105, or more favorably 102-104 PFU/dose, and inoculated into napes of one day of age chickens subcutaneously or into embryonated eggs by syringe or by any apparatus for injection.
The avian polyvalent vaccine gives chickens at least 50% protection against the challenge with a variety of different subtypes of IBDV. Different subtypes mean the combination of two or more subtypes of IBDV selected from at least two, or more favorably from three, or more favorably from all subtypes of IBDV. The combination of three strains, STC belonging to the classical virulent subtype, Delaware E and AL-2 belonging to the variant subtype, is an example.
In the present invention, the protection against the IBDV challenge is determined by the ratio of protected birds to total tested birds. Essentially, the vaccinated chickens are challenged intraocularly with 103 EID50/dose or more of IBDV and necropsied one week later to detect any notable lesions. Protected birds without notable lesions have (1) the weight ratio of the bursa of Fabricius to the body (B/B ratio) which is not statistically different from that of non-vaccinated, non-challenged chickens, and show (2) no malformation of the Bursa of Fabricius such as edematization, hemorrhage, yellowish exudate, discoloration, atrophy, or gelatinous exudate. The challenge testing is valid only when all chickens of the non-vaccinated and challenged control show the notable legions indicating 0% protection.